An exploratory study for the discovery of non-invasive hepatocellular carcinoma biomarkers among high-risk hepatitis C virus infected patients

Hepatocellular Carcinoma (HCC) is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. Chronic infections with Hepatitis B virus (HBV) and/or Hepatitis C virus (HCV) are the major risk factors for the development of HCC. The incidence of HBV -associated HCC is in decline as a result of an effective HBV vaccine; however, since an equally effective HCV vaccine has not yet been developed, there are 130 million HCV infected patients worldwide who are at a high-risk for developing HCC. Because reliable parameters and/or tools for the early detection of HCC among high-risk individuals are severely lacking, HCC patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Using urine as a non-invasive sample source, two different approaches (proteomic-based and genomic-based approaches) were pursued with the common goal of discovering potential biomarker candidates for the early detection of HCC among high-risk chronic HCV infected patients. Urine was collected from 106 HCV infected Egyptian patients, 32 of whom had already developed HCC and 74 patients who were diagnosed as HCC-free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins, Trans-renal nucleic acid (Tr-NA) and microRNA (miRNA) were isolated from urine using novel methodologies and silicon carbide-loaded spin columns. In the first, "proteomic-based", approach, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify potential candidates from pooled urine samples. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR (qRT-PCR). This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and 11 Moemen Abdalla HCC Biomarkers Heat Shock Protein 60 (HSP60), were characteristic events among HCC-post HCV infected patients. As a single-based HCC biomarker, CAF-1 over-expression identified HCC among HCV infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-lIHSP60 tandem identified HCC among HCV infected patients with a specificity of 92%, sensitivity of 61 % and with an overall diagnostic accuracy of 77%. In the second genomic-based approach, two different approaches were processed. The first approach was the miRNA-based approach. The expression levels of miRNAs isolated from urine were studied using the Illumina MicroRNA Expression Profiling Assay. This was followed by qRT-PCR-based validation of deregulated expression of identified miRNA candidates among all the patients. This approach shed the light on the deregulated expression of a number of miRNAs, which may have a role in either the development of HCC among HCV infected patients (i.e. miR-640, miR-765, miR-200a, miR-521 and miR-520) or may allow for a better understanding of the viral-host interaction (miR-152, miR-486, miR-219, miR452, miR-425, miR-154 and miR-31). Moreover, the deregulated expression of both miR-618 and miR-650 appeared to be a common event among HCC-post HCV infected patients. The results of the search for putative targets of these two miRNA suggested that miR-618 may be a potent oncogene, as it targets the tumor-suppressor gene Low density lipoprotein-related protein 12 (LPR12), while miR-650 may be a potent tumor-suppressor gene, as it is supposed to downregulate the TNF receptor-associated factor-4 (TRAF4) oncogene. The specificity of miR-618 and miR-650 deregulated expression patterns for the early detection of HCC among HCV infected patients was 68% and 58%, respectively, whereas the sensitivity was 64% and 72%, respectively. When the deregulated expression of both miRNAs was combined as a tandem biomarker, the specificity and the sensitivity were 75% and 58% respectively. 111 Moemen Abdalla HCC Biomarkers In the second, "Trans-renal nucleic acid-based", approach, the urinary apoptotic nucleic acid (uaNA) levels of 70ng/mL or more were found to be a good predictor of HCC among chronic HCV infected patients. The specificity and the sensitivity of this diagnostic approach were 76% and 86%, respectively, with an overall diagnostic value of 81 %. The uaNA levels positively correlated to HCC disease progression as monitored by epigenetic changes of a panel of eight tumor-suppressor genes (TSGs) using methylation-sensitive PCR. Moreover, the pairing of high uaNA levels (:::: 70 ng/mL) and CAF-1 over-expreSSIOn produced a highly specific (l 00%) multiple-based HCC biomarker with an acceptable sensitivity of 64%, and with a diagnostic accuracy of 82%. In comparison to the previous pairing, the uaNA levels (:::: 70 ng/mL) in tandem with HSP60 over-expression was less specific (89%) but highly sensitive (72%), resulting in a diagnostic accuracy of 64%. The specificities of miR-650 deregulated expression in combination with either high uaNA content or HSP 60 over-expression were 82% and 79%, respectively, whereas, the sensitivities of these combinations were 64% and 58%, respectively. The potential biomarkers identified in this study compare favorably with the diagnostic accuracy of the a-fetoprotein levels test, which has a specificity of 75%, sensitivity of 68% and an overall diagnostic accuracy of 70%. Here we present an intriguing study which shows the significance of using urine as a noninvasive sample source for the identification of promising HCC biomarkers. We have also introduced new techniques for the isolation of different urinary macromolecules, especially miRNA, from urine. Furthermore, we strongly recommend the potential biomarkers indentified in this study as focal points of any future research on HCC diagnosis. A larger testing pool will determine if their use is practical for mass population screening. This explorative study identified potential targets that merit further investigation for the development of diagnostically accurate biomarkers isolated from 1-2 mL urine samples that were acquired in a non-invasive manner.

Simultaneous extraction of order parameters and orientational distribution fuctions from 31[superscript]P NMR spectra of magnetically partially oriented phospholipid bilayers

Order parameter profiles extracted from the NMR spectra of model membranes are a valuable source of information about their structure and molecular motions. To al1alyze powder spectra the de-Pake-ing (numerical deconvolution) ~echnique can be used, but it assumes a random (spherical) dist.ribution of orientations in the sample. Multilamellar vesicles are known to deform and orient in the strong magnetic fields of NMR magnets, producing non-spherical orientation distributions. A recently developed technique for simultaneously extracting the anisotropies of the system as well as the orientation distributions is applied to the analysis of partially magnetically oriented 31p NMR spectra of phospholipids. A mixture of synthetic lipids, POPE and POPG, is analyzed to measure distortion of multilamellar vesicles in a magnetic field. In the analysis three models describing the shape of the distorted vesicles are examined. Ellipsoids of rotation with a semiaxis ratio of about 1.14 are found to provide a good approximation of the shape of the distorted vesicles. This is in reasonable agreement with published experimental work. All three models yield clearly non-spherical orientational distributions, as well as a precise measure of the anisotropy of the chemical shift. Noise in the experimental data prevented the analysis from concluding which of the three models is the best approximation. A discretization scheme for finding stability in the algorithm is outlined

The Military Monitor and American Register- 31 August 1812

The Military Monitor was a weekly periodical that was published every Monday. The first issue was printed for August 17, 1812 and is believed to have ceased in 1814, as the last issue located was April 2, 1814. The periodical was suspended with the November 23, 1812 issue and resumed with the December 14, 1812 issue. The quote at the top of the first page is "The public good our end". The periodical's various authors included: Desnoues, Joseph, 1794?-1837. O'Connor, Thomas, 1770-1855. Hardcastle, John, 1778?-1835. Van Pelt, Peter, 1779?-1843. Wall, Stephen. Van Riper, Nicholas. Other authors are believed to be the American Antiquarian Society. Proprietors: T. O'Connor and S. Wall, 1812; T. O'Connor, 1812- . Printers: Hardcastle and Van Pelt, for T. O'Connor and S. Wall, Sept. 14-Oct. 5, 1812; J. Desnoues, Oct. 12, 1812- ; N. Van Riper, Nov. 6, 1813- . This issue was included in a bound volume of the Military Monitor and American Register. Other Dates included are: 1812 October 12 1812 October 19 1812 November 23 1812 December 14 1812 December 21 1813 January 11 1813 February 1 1813 March 29 1813 April 5 1813 April 12 1813 April 26 1813 May 31

The Military Monitor and American Register- 31 May 1813

The Military Monitor was a weekly periodical that was published every Monday. The first issue was printed for August 17, 1812 and is believed to have ceased in 1814, as the last issue located was April 2, 1814. The periodical was suspended with the November 23, 1812 issue and resumed with the December 14, 1812 issue. The quote at the top of the first page is "The public good our end". The periodical's various authors included: Desnoues, Joseph, 1794?-1837. O'Connor, Thomas, 1770-1855. Hardcastle, John, 1778?-1835. Van Pelt, Peter, 1779?-1843. Wall, Stephen. Van Riper, Nicholas. Other authors are believed to be the American Antiquarian Society. Proprietors: T. O'Connor and S. Wall, 1812; T. O'Connor, 1812- . Printers: Hardcastle and Van Pelt, for T. O'Connor and S. Wall, Sept. 14-Oct. 5, 1812; J. Desnoues, Oct. 12, 1812- ; N. Van Riper, Nov. 6, 1813- . This issue was included in a bound volume of the Military Monitor and American Register. Other Dates included are: 1812 August 31 1812 October 12 1812 October 19 1812 November 23 1812 December 14 1812 December 21 1813 January 11 1813 February 1 1813 March 29 1813 April 5 1813 April 12 1813 April 26

The New York Weekly Museum Newspaper, Vol. I, No.26- October 31, 1812

A weekly paper that was published from 1805 to 1814. War related news includes: Page 103 - Weekly Retrospect: Troop movements of Captain Morgan and a company of light dragoons from Niagara to Greenbush; report on the Battle of Queenston Heights including a casualty listing and a mention of the death of Major General Isaac Brock and Colonel McDonald and the American's respect for Brock; British attach of Fort Erie and the Village of Black Rock; the privateer Globe under Captain Murphy returning to Baltimore after capturing a British letter of marque; First Nation / Indian attacks near St. Louis; movements of General W. Harrison and army from Fort Defiance to the rapids of Miama (Miami). The rest of the newspaper contains literary works (poems and translations), essays (including on celibacy), marriages, deaths and other anecdotes. The motto on the front page states: "Visiting Every Flower with Labour Meet, and Gathering all its Treasures, Sweet by Sweet."

Letter - Colonel Albert D. Shaw to Francis Lynde Stetson, 31 March 1892

A letter from Colonel Albert D. Shaw to Francis Lynde Stetson dated March 31, 1892. The letter is in regards to correspondence with the Attorney General in efforts to expedite the passing of an Act through Ontario Parliament. The act was introduced a week later (April 6, 1892) confirming the agreement between the Queen Vctoria Niagara Falls Park and the Canadian Niagara Power Company.

Portsmouth Oracle, 23 April 1814 (vol. 25, no. 31) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

Portsmouth Oracle, 31 December 1814 (vol. 25, no. 67) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

American Mercury, 16 August 1814 (vol. 31, 1572) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812. The 1814 Aug. issues report events of the Battle of Chippewa.

The News, 31 October 1813 (no. 434) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

The War, 31 October 1812 (vol. 1, no. 19) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

The War, 31 May 1814 (vol. 2, no. 50, whole no. 102) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.